摘要
目的通过Pitx2-Morpholino反义寡核苷酸显微注射方法沉默斑马鱼Pitx2基因,研究Pitx2基因早期功能下调对斑马鱼牙齿发育相关基因的影响。方法通过Morpholino反义寡核苷酸显微注射方法功能下调斑马鱼Pitx2基因,观察胚胎在72 h内的发育情况。并利用RT-PCR技术直接克隆Bmp4、Fgf8特异性基因片段,制作针对Bmp4、Fgf8的基因探针,对斑马鱼发育早期多个时段的胚胎进行整体原位杂交,观察Pitx2基因下调对Bmp4、Fgf8在斑马鱼腮弓表达的影响。结果成功合成Bmp4、Fgf8基因探针。显微镜下观察实验斑马鱼胚胎在72 h内的生长发育情况,发现经Morpholino显微注射斑马鱼胚胎出现发育迟缓或停止、心包水肿、脊索畸形、眼睛发育异常等形态学改变。原位杂交发现相对于野生型,Morpholino显微注射斑马鱼腮弓处Bmp4基因表达有所增强,Fgf8基因表达有所减弱。结论 Pitx2基因功能下调可使斑马鱼早期发育发生形态改变并使Bmp4基因表达增强,Fgf8基因表达减弱。
Objective The aim of this study was to silence Pitx2 gene by Pitx2- morpholino oligos microinjection so as to explore the effect of down-regulated Pitx2 on tooth development related genes in zebrafish. Methods Pitx2 gene was down-regulated by mor- pholino oligos microinjection. We observed the development of the embryos in 72 hours with microscopes, synthesized gene probes for Bmp4,F8 by cloning specific gene fragment of Bmp4 and Fgf8 by RT-PCR directly,and then carried out the in situ hybridization with embryos from different stages during early dental development to observe the effect of down-regulated Pitx2 on expression of Bmp4 and Fgf8 in zebrafish branchial arch. Results We successfully synthesized gene probes for Bmp4 and FgfS, and observed the development of the embryos in 72 hours with microscopes. Growth retardation or termination, pericardial edema, spinal and eyes deformity appeared in the zebrafish embryos in Morpholino groups after Pitx2- morpholino oligos microinjection. In situ hybridization showed that down-regulated Pitx2 increased the expression of Bmp4, and repressed the expression of Fgf8 in branchial arch of zerafish after Pitx2morpholino oligos microinjection,compared with wild zebrafish. Conclusions Down-regulated Pitx2 made zebrafish growing with retardation or yermination, and increase the expression of Bmp4 and repressed the expression of FgfS.
出处
《口腔医学》
CAS
2013年第2期78-83,共6页
Stomatology
基金
江苏省科技厅自然科学基金项目(BK2010528)
江苏高校优势学科建设工程资助项目
